The present invention relates to a method for the treatment of contaminated substances such as wastewaters and soils to remove organic contaminates and/or heavy metals using soybean peroxidase (SBP), and a peroxide.
Existing methods for the removal of organic and inorganic pollutants from contaminated sources include adsorption, extraction, microbial and chemical oxidation, electrochemical techniques and irradiation. However, all of these methods suffer from serious shortcomings such as high costs, incompleteness of purification, formation of hazardous byproducts and low efficiency. see, e.g., M. W. Slein et al, Degradation of Chemical Carcinogens, Van Nostrand Reinhold Co., 1980; Cleaning our Environment--A Chemical Perspective, American Chemical Society, 1978. Therefore, alternative methods of removing aromatic compounds from wastewater are highly desirable.
The use of peroxidase enzymes to remove hazardous aromatic materials from wastewaters is well known. For example, Alberti and Klibanov, "Peroxidase for removal of Hazardous Aromatics form Industrial Wastewaters," Biological Detoxication, Chapter 22, 349-356 (1982) disclose that phenols and aromatic amines can be removed from wastewaters as high molecular weight polymers by the action of horseradish peroxidase enzymes on such aromatic compounds. The disclosed method relies on the ability to catalyze, with hydrogen peroxide, the oxidation of a variety of phenols and aromatic amines. Phenolic and aromatic amine-free radicals are generated and these free radicals diffuse from the active center of the enzyme into solution where they polymerize to form high molecular weight polyaromatic products which are insoluble and can be readily separated from the water by, e.g., filtration.
U.S. Pat. No. 4,623,465 to Klibanov discloses a method for removing aromatic substances such as phenols and aromatic amines from aqueous solutions by adding chemicals such as horseradish peroxidase enzyme and peroxide substrates to such solutions which contain two or more different aromatic substances. According to Klibanov '465, phenols are removed from water with about 75% efficiency by horseradish peroxidase and peroxide at a pH of 5.5 at room temperature. However, when another aromatic compound such as orthodianisidine, benzidine or 8-hydroxyquinoline is added to the phenol, then the phenol and the other aromatic substance are both removed form the solution with better than 99.5% efficiency.
U.S. Pat. No. 4,478,683 to Orndorff describes a method for enzymatically killing and controlling the growth of microorganisms in industrial wastewater streams by the catalytic oxidation of monophenolic compounds added to or naturally occurring in the stream. The preferred enzyme is a plant peroxidase, e.g., from horseradish, turnip, etc.
Shannon et al., "immobilization of Leachable Toxic Soil Pollutants", Appl. Env. Microbiol., 54, No. 7, 1719-1723, (1988) discuss the use of horseradish peroxidase to immobilize phenolic pollutants in sand and soil columns. The immobilized pollutants were non-leachable from the sand and soil for extended periods of time.
Because of the wide use of chlorine as a bleaching agent for chemically produced wood pulps in the pulp and paper industry, dilute pulp mill bleaching effluents contain undesirable levels of adsorbable organic halogens (AOX) in the form of chlorophenols, chloroaliphatics, chlorocatechols, polymerized chloroaromatics, etc. Also, chlorophenols are major intermediates of phenoxyalkanoate herbicides and other pesticides which retain their toxic properties for an indefinite period of time since they easily form soil-bound residues. The effect of chlorinated compounds such as pentachlorinatedphenol (PCP), polychlorinated biphenyls (PCB), chlorinated benzene, etc. released into the environment is an immediate concern of the entire population, and increasingly stringent government standards are requiring that the total levels of such chlorinated compounds released into the environment be substantially reduced.
Roy-Arcand and Archibald, "Direct Dechlorination of Chlorophenolic Compounds by Laccases form Trametes (Coriolus) Versicolor", Enzyme Microb. Technol., Vol. 13, pp. 194-203 (1991) disclose that horseradish peroxidase will dechlorinate chlorinated compounds present in pulp mill bleach plant effluents. The authors suggest that the enzymatic treatment of such wastewaters using horseradish peroxidase offers a potentially superior method of wastewater treatment.
Ferrer et al. "Decolorization of Kraft Effluent by Free and Immobilized Lignin Peroxidase and Horseradish Peroxidase", Biotechnology Letters, Vol. 13, No. 3, 577, 582 (1991) disclose that in the free state some enzymes such as lignin peroxidase and horseradish peroxidase will remove color from kraft effluents but in all cases studied, the immobilized enzymes were considerably more efficient than the enzyme in the free form.
Wigfield et al, "Kinetics and Mechanisms of Oxidation of Mercury by Peroxidase", Can. J. Chem., Vol. 63, 2940-2944, (1985) reported the kinetics of the oxidation of zero-valent mercury by horseradish peroxidase.
However, in the past, peroxidase enzymes have not been available at a cost and in a purity amenable to many biocatalytic processes such as for the treatment of contaminated waters, and particularly wastewater treatment. For example, horseradish roots, a common source of horseradish peroxidase (HRP), are cultivated generally in small quantities and are propagated through root cuttings, thus making it difficult to scale up production. The limited availability of the horseradish root extract coupled with the shortage of alternative sources of enzyme has created a very expensive market for such enzymes. Accordingly, there exists in the marketplace a need for an abundant and relatively inexpensive source of peroxidase for use in treating contaminated waters, and particularly for use in wastewater treatment to remove hazardous or toxic materials.